Bottom Line:
A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size.Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1.Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

Affiliation: Department of Biochemistry and Biophysics, The Hormone Research Institute, University of California, San Francisco, California 94143-0534, USA.

ABSTRACTCarrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

Figure 11: Cytosolic clathrin and COPI are not needed for the cell-free budding of SV. (a) Normal rat brain cytosol was immunodepleted of clathrin heavy chains using the X22 mAb bound to protein G–Sepharose. The extent of depletion was confirmed by immunoblotting equal amounts of rat brain cytosol protein before (lane 1) or after (lane 2) the immunoabsorption with the TD.1 anti-clathrin antibody. Lane 3 corresponds to clathrin heavy chains retained on the beads. (b) In vitro reaction mixtures were prepared containing labeled N49A/PC12 cell homogenates, ATP regenerating system in the presence of normal or clathrin- depleted rat brain cytosol. Reactions containing normal rat brain cytosol (supplemented with either 11–22 μg/ml of affinity-purified anti–β-COP antipeptide antibody EAGE, 1 μM of either GST-WBP1-SS or GST-WBP1-KK, or a combination of EAGE antibody plus GST-WBP1-KK) were kept for at least 3 h at 4°C before warming to 37°C. None of the treatments substantially modified the budding of SV.

Mentions:
ARF molecules have been implicated in recruiting coats made up either of coatomers or of adaptor/clathrin complexes. To test if clathrin was involved in the budding process, it was quantitatively removed from rat brain cytosol using mAb to clathrin (Fig. 11 a). No significant reduction in budding efficiency was observed (Fig. 11 b). To examine the involvement of COPI coatomers in vesicle formation, we used conditions that are known to inhibit COPI function during endosomal sorting (Whitney et al., 1995) or binding of COPI to membranes (Lowe and Kreis, 1995). Addition of antibodies to β-COP, a fusion protein corresponding to the KKXX signal motif of WBPI (GST-KK) (Cosson and Letourneur, 1994), or a combination of the antibody and the inhibitory peptide had no effect on vesicle formation.

Figure 11: Cytosolic clathrin and COPI are not needed for the cell-free budding of SV. (a) Normal rat brain cytosol was immunodepleted of clathrin heavy chains using the X22 mAb bound to protein G–Sepharose. The extent of depletion was confirmed by immunoblotting equal amounts of rat brain cytosol protein before (lane 1) or after (lane 2) the immunoabsorption with the TD.1 anti-clathrin antibody. Lane 3 corresponds to clathrin heavy chains retained on the beads. (b) In vitro reaction mixtures were prepared containing labeled N49A/PC12 cell homogenates, ATP regenerating system in the presence of normal or clathrin- depleted rat brain cytosol. Reactions containing normal rat brain cytosol (supplemented with either 11–22 μg/ml of affinity-purified anti–β-COP antipeptide antibody EAGE, 1 μM of either GST-WBP1-SS or GST-WBP1-KK, or a combination of EAGE antibody plus GST-WBP1-KK) were kept for at least 3 h at 4°C before warming to 37°C. None of the treatments substantially modified the budding of SV.

Mentions:
ARF molecules have been implicated in recruiting coats made up either of coatomers or of adaptor/clathrin complexes. To test if clathrin was involved in the budding process, it was quantitatively removed from rat brain cytosol using mAb to clathrin (Fig. 11 a). No significant reduction in budding efficiency was observed (Fig. 11 b). To examine the involvement of COPI coatomers in vesicle formation, we used conditions that are known to inhibit COPI function during endosomal sorting (Whitney et al., 1995) or binding of COPI to membranes (Lowe and Kreis, 1995). Addition of antibodies to β-COP, a fusion protein corresponding to the KKXX signal motif of WBPI (GST-KK) (Cosson and Letourneur, 1994), or a combination of the antibody and the inhibitory peptide had no effect on vesicle formation.

Bottom Line:
A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size.Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1.Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.

Affiliation:
Department of Biochemistry and Biophysics, The Hormone Research Institute, University of California, San Francisco, California 94143-0534, USA.

ABSTRACTCarrier vesicle generation from donor membranes typically progresses through a GTP-dependent recruitment of coats to membranes. Here we explore the role of ADP ribosylation factor (ARF) 1, one of the GTP-binding proteins that recruit coats, in the production of neuroendocrine synaptic vesicles (SVs) from PC12 cell membranes. Brefeldin A (BFA) strongly and reversibly inhibited SV formation in vivo in three different PC12 cell lines expressing vesicle-associated membrane protein-T Antigen derivatives. Other membrane traffic events remained unaffected by the drug, and the BFA effects were not mimicked by drugs known to interfere with formation of other classes of vesicles. The involvement of ARF proteins in the budding of SVs was addressed in a cell-free reconstitution system (Desnos, C., L. Clift-O'Grady, and R.B. Kelly. 1995. J. Cell Biol. 130:1041-1049). A peptide spanning the effector domain of human ARF1 (2-17) and recombinant ARF1 mutated in its GTPase activity, both inhibited the formation of SVs of the correct size. During in vitro incubation in the presence of the mutant ARFs, the labeled precursor membranes acquired different densities, suggesting that the two ARF mutations block at different biosynthetic steps. Cell-free SV formation in the presence of a high molecular weight, ARF-depleted fraction from brain cytosol was significantly enhanced by the addition of recombinant myristoylated native ARF1. Thus, the generation of SVs from PC12 cell membranes requires ARF and uses its GTPase activity, probably to regulate coating phenomena.